To regulate their functions, enzymes may be modified covalently. For example, protein phosphorylation is a modification central to the regulation of metabolism and other cell functions. Another modification is ADP-ribosylation, best known from the example of guanine nucleotide- binding regulatory proteins that are ADP-ribosylated by the bacterial enzymes cholera toxin and pertussis toxin. Animal cells contain enzymes that catalyze ADP-ribosylation reactions like those of the bacterial toxins, and the goal remains to determine how endogenous ADP-ribosylation pathways are regulated, and how endogenous ADP-ribosylation regulates events in the cell. One possible regulatory connection is the recently described role of nitric oxide (NO), a biological messenger, in endogenous ADP-ribosylation reactions. The protein glyceraldehyde-3- phosphate dehydrogenase (GAPDH) is modified in reactions with NAD and NO, in a process presumed to be ADP-ribosylation. In our work, the modification of GAPDH was investigated by reacting the enzyme with NO and three species of NAD radiolabeled in different positions. In these reactions, radiolabel was incorporated into GAPDH equally with nicotinamide- and adenine-labeled NAD, indicating that the modification of GAPDH was not ADP-ribosylation, but was binding of the entire NAD molecule. Though GAPDH was modified with NAD to an extent of only 1-2%, under the same conditions, GAPDH activity was inhibited by 60-80%. This comparison indicates that most of the inhibition was not due to covalent modification with NAD, but was probably due to the reported S- nitrosylation of the enzyme active site, which occurs at stoichiometric levels. Other enzymes were examined for NO-stimulated modification with NAD; only alcohol dehydrogenase of nine other enzymes tested was modified, and at <10% of the level of GAPDH modification. Aldehyde dehydrogenase was inhibited by NO to the same extent as GAPDH, but was not modified at all with NAD. The modification of GAPDH with NAD represents an unexpected, altered chemical reactivity of a nitrosylated protein, and likely explains the "ADP-ribosylation" reactions reported to occur in the presence of NO.